Peptides with affinity for a phospholipid and uses

ABSTRACT

The present invention relates to a peptide for the specific recognition of lipid vectors. The peptide of the invention comprises the peptide sequence (I; SEQ ID NO: 15) below:  
                     (I)         J 1 -J 2 -J 3 -J 4 -J 5 -J 6 -Z 7 -U 8 -J 9 -J 10 -U 11 -Arg-J 13 -J 14 -           U 15 -Lys-Gly-X 18 -Gly-Thr-J 21 -Glu-J 23 -J 24 -U 25 -J 26 -           J 27 -J 28 -U 29 -J 30 -J 31 -Arg-J 33 -J 34 -J 35 -J 36 -B 37 -J 38 -           J 39 -U 40 -J 41 -J 42 -J 43 -U 44 -J 45 -J 46 -J 47 -J 48 -J 49 -Arg-           J 51 -U 52 -J 53 -J 54 -Asp-U 56 -Lys-Ser-Z 59 -Leu-J 61 -J 62 -           J 63 -J 64 -Z 65 -J 66 -J 67 -U 68 -J 69 -J 70 -J 71 -U 72 -J 73 -J 74 -           J 75                             
 
in which the amino acids J are chosen, independently of one another, from essential amino acids, or derivatives thereof, such that at least 50% of them are polar residues chosen from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Lys, Orn, Pro, Ser, Thr and Tyr; the amino acids U are chosen, independently of one another, from Ala, Cys, Gly, Ile, Leu, Met, Phe, Trp, Tyr and Val; the amino acid X 18  is chosen, independently of the other amino acids of the sequence, from Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Ser, Thr, Trp, Tyr and Val; the amino acid B 37  is chosen, independently of the other amino acids of the sequence, from Arg, Ala, Cys, Gly, Ile, Leu, Met, Phe, Trp, Tyr and Val; the amino acid Z 7  is chosen, independently of the other amino acids of the sequence, from Asp and Gly; the amino acids Z 59  and Z 65  are chosen from Glu, Asp, Lys or Arg; and the superscripts of the residues J, Z, U, X and B represent the position of these amino acids in said sequence.

TECHNICAL FIELD

The present invention relates to a family of peptides with affinity fora phospholipid and also to various uses of this peptide, in particularin the pharmaceutical field.

In general, the peptides of the present invention are useful for thespecific recognition of lipid molecules. They can be used forengineering and creating compounds that recognize and sequester thelipids, in particular negatively charged lipids, such asphosphatidylserines, phosphatidic and lysophosphatidic acids,phosphatidyl-glycerols, cardiolipins and sphingosine-1-phosphates.

The abovementioned lipids play an important role in particular in cellsignalling and may be present at the outer surface of cell membranesand/or may circulate in the blood subsequent to very diversepathological events.

Various cellular events result in the appearance of negatively chargedlipids, and in particular phosphatidylserines (PS), at the outer surfaceof cells; these events can result either from a fortuitous orpathological alteration of the cell, or from a programmed cell eventsuch as cell death or apoptosis. The appearance of PS at the outersurface of cells therefore constitutes an important “primary message”reflecting the existence of a dysfunction. In the case of the bloodclotting process, the mechanism is well described: the alteration in theblood vessel endothelial cells, either for accidental reasons or formore complex pathological reasons, brings about the appearance of thisPS message at the outer surface of the cells in contact with the bloodenvironment. This message is immediately recognized by certaincirculating proteins which then trigger a cascade of events resulting inthe well known phenomenon of blood clotting.

The invention takes advantage of the property of the peptides that itprovides of binding, in the presence or absence of calcium, to lipidsand in particular to those which are negatively charged, for developingcompounds that can be used as research, diagnostic and therapeutic toolsin the field of lipid effector recognition and of the detection ofapoptosis, of blood clotting disorders, of septic shock and acuteinflammatory pathologies in particular.

As regards research and diagnosis, the peptides of the invention can,for example, be coupled to molecules for detection, for example to afluorescent molecule, to one of the partners of the avidin-biotinsystem, to a radio element with a short life, to a paramagneticcompound, or to particles of gold or of dense compounds for electronmicroscopy. With these molecules for detection, it is possible, forexample, to detect apoptotic cells or to recognize negatively chargedmembrane microdomains.

The peptides of the present invention can therefore be used for “invitro” detection of pathologies involving the appearance of negativecharges at the surface of cells and the release of microvesicules intothe blood.

The peptides of the present invention can also be used for the in vivodetection and the imaging of apoptotic foci, of thrombotic zones and, ingeneral, of any centre exposing negatively charged lipids, when thesepeptides are coupled, for example, to a radio element with a shortlifetime (scintigraphic images acquired by Single Photon EmissionComputed Tomography (SPECT) or by Positron Emission Tomography (PET)) orto any contrast compound such as a gadolinium complex for magneticresonance imaging (MRI).

As regards therapy, in general, the peptides of the present inventioncan be used alone or coupled to a therapeutic molecule for preparing amedicinal product. Such a medicinal product can, for example, be usedfor targeting this molecule to zones exhibiting negative charges, suchas tumours exhibiting foci of apoptotic cells or inflammatory tumours.

The peptides of the present invention can, for example, be coupled tomolecules with a thrombolytic action, for preparing a medicinal productthat can be used in the treatment and the prophylaxis of thrombosis, orfor preparing a molecule covering all thrombogenic biomaterials. Thepeptides of the present invention can therefore be used for targetingthrombolytic molecules to the site of the thrombus or to thrombogeniczones.

In another example of application of the present invention, the peptidesof the invention can be used alone or coupled to an anti-inflammatorymolecule, for preparing a medicinal product that can be used, forexample, in acute pathologies such as asthma, ulcerative colitis (UC),Crohn's disease, septic shock, collagen diseases and arthritis.

Other applications will become further apparent to those skilled in theart on reading the description which follows.

STATE OF THE ART

A family of proteins, called annexins, have been described in the priorart as exhibiting reversible functional anchoring to the cell membrane,regulated by the calcium concentration and the presence of anionicphospholipids. The annexins constitute a family of proteins expressed invery diverse tissues, both in animals and in plants. It appears thatthey are expressed neither in bacteria nor in yeast.

The structure of annexins comprises four domains of approximately 70amino acids, or residues, which are very moderately homologous in termsof sequence but virtually identical in terms of topology.

In document WO 92/19279, J. Tait describes conjugates with affinity forphospholipids. He describes in particular the use of an annexin, inparticular of annexin V, for producing an active conjugate that can beused as a thrombolytic agent.

Unfortunately, the compound described in that document and prepared fromthe whole annexin by means of a process of genetic recombination hasmany drawbacks, which are in particular a low yield and a highproduction cost. The major drawbacks are especially the fact that afragile conjugate is obtained due to its complex topology resulting inirreversible unfolding. In addition, these molecules exhibit greattoxicity for the kidney and the heart.

The present inventors have described, in application WO-A-00/20453, afirst family of peptides that overcomes the abovementioned drawbacks andhas affinity for phospholipids and improved stability.

DISCLOSURE OF THE INVENTION

The aim of the present invention is to provide a novel family ofpeptides with affinity for lipids, in particular for phospholipids, thatis more specific and further improved with respect to the products ofthe prior art.

The peptides of the invention also have the advantages of being morechemically stable than the compounds of the prior art and of being ableto be produced reproducibly, with a high yield and a very low productioncost compared with the compounds of the prior art.

The peptides of the present invention are characterized in that theycomprise the peptide sequence (I) below: (I)J¹-J²-J³-J⁴-J⁵-J⁶-Z⁷-U⁸-J⁹-J¹⁰-U¹¹-Arg-J¹³-J¹⁴-U¹⁵-Lys-Gly-X¹⁸-Gly-Thr-J²¹-Glu-J²³-J²⁴-U²⁵-J²⁶-J²⁷-J²⁸-U²⁹-J³⁰-J³¹-Arg-J³³-J³⁴-J³⁵-J³⁶-B³⁷-J³⁸-J³⁹-U⁴⁰-J⁴¹-J⁴²-J⁴³-U⁴⁴-J⁴⁵-J⁴⁶-J⁴⁷-J⁴⁸-J⁴⁹-Arg-J⁵¹-U⁵²-J⁵³-J⁵⁴-Asp-U⁵⁶-Lys-Ser-Z⁵⁹-Leu-J⁶¹-J⁶²-J⁶³-J⁶⁴-Z⁶⁵-J⁶⁶-J⁶⁷-U⁶⁸-J⁶⁹-J⁷⁰-J⁷¹-U⁷²-J⁷³-J⁷⁴- J⁷⁵in which J, Z, U, X and B represent amino acids such that:

-   -   the amino acids J are chosen, independently of one another, from        natural amino acids or derivatives thereof, such that at least        50% of them are polar residues chosen from Arg, Asn, Asp, Cys,        Gln, Glu, Gly, His, Lys, Orn, Pro, Ser, Thr and Tyr,    -   the amino acids U are chosen from Ala, Cys, Gly, Ile, Leu, Met,        Phe, Trp, Tyr and Val,    -   the amino acid X¹⁸ is chosen, independently of the other amino        acids of the sequence, from Ala, Asn, Cys, Gln, Gly, His, Ile,        Leu, Met, Phe, Ser, Thr, Trp, Tyr and Val,    -   the amino acid B³⁷ is chosen, independently of the other amino        acids of the sequence, from Arg, Ala, Cys, Gly, Ile, Leu, Met,        Phe, Trp, Tyr and Val,    -   the amino acid Z⁷ is chosen, independently of the other amino        acids of the sequence, from Asp and Glu,    -   the amino acids Z⁵⁹ and Z⁶⁵ are, independently, Glu, Asp, Lys or        Arg,        the superscripts of J, Z, U, X and B representing the position        of these amino acids in said sequence.

The peptide sequence above folds up in space so as to adopt its tertiaryconformation, which is the active form of the peptide.

Amino acids 12, 15, 16, 17, 19, 20, 22, 50, 55, 57, 58, 59, 60 and 65are the amino acids, or residues, of the present invention that aredirectly or indirectly involved in the binding to lipids, i.e. they areinvolved either in the three-dimensional structure of the peptide sothat it adopts its active conformation allowing recognition of anegatively charged lipid, or in the peptide recognition site.

The amino acids J are the surface amino acids, or residues, of thispeptide when it is in its folded and active conformation. These residuesare arranged spatially such that they are partially or completelyexposed to the solvent. According to the present invention, these aminoacids J may, for example, be chosen, independently of one another, fromall the natural amino acid residues Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Orn, Phe, Pro, Ser, Thr, Trp, Tyr and Val,and such that at least 50% of them are polar residues chosen from Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Lys, Orn, Pro, Ser and Thr. Examplesare given in the sequence listing in the appendix.

The amino acids U are the core residues of this peptide. In the foldedand active conformation of the peptide, they are spatially arrangedclose to one another and are not exposed to the solvent. They constitutethe hydrophobic core of the protein. The compact assembly of the atomsof these residues plays a predominant role in the stability of thepeptide in its active conformation. These residues can be chosen fromthe list of amino acids U described above. Various examples ofcombinations of core residues in the peptide of sequence (I) of thepresent invention are given in table (1) below: TABLE 1 U⁸ U¹¹ U¹⁵ U²⁵U²⁹ B³⁷ U⁴⁰ U⁴⁴ U⁵² U⁵⁶ U⁶⁸ U⁷² Ex a) Val Leu Met Ile Leu Arg Ile TyrLeu Leu Val Leu Ex b) Ala Ile Ile Ile Leu Arg Ile Tyr Leu Leu Ile Leu Exc) Ala Ile Ile Ile Leu Arg Ile Tyr Leu Leu Met Val Ex d) Ala Leu Met LeuLeu Arg Ile Tyr Leu Leu Ile Met Ex e) Ala Leu Met Ile Ile Arg Val TyrLeu Leu Ile Met Ex f) Ala Leu Met Ile Ile Arg Ile Phe Leu Leu Ile Met Exg) Ala Leu Met Ile Val Arg Ile Phe Leu Leu Ile Phe Ex h) Val Leu Met IleLeu Arg Ile Phe Leu Leu Ile Met Ex i) Ala Leu Met Ile Leu Arg Ile PheLeu Leu Ile Met Ex j) Ala Leu Met Ile Leu Arg Ile Tyr Leu Leu Ala Ala Exk) Val Leu Met Ile Leu Arg Ile Tyr Leu Leu Val Leu Ex l) Val Leu Met IleLeu Arg Ile Phe Leu Leu Val Leu(Ex = example)

The function of the residue X¹⁸ is to maintain the structure of theGly-X-Gly loop in the active form of the peptide, in particular wherethe residues Z⁵⁹ and Z⁶⁵ are Glu, to modulate the hydrophobic andlipophilic nature of this loop, and to optionally provide new specificinteractions with phospholipids. This is the case, for example, of theresidues Asn, Cys, Ser, Thr, Trp and Tyr.

The residues Z⁵⁹ and Z⁶⁵ may advantageously be lysine residues, theeffect of which is to replace the calcium ion with the positivelycharged —NH₃ ⁺ group of the lysine and to improve the affinity of thepeptide for a negatively charged membrane.

The peptide (I) of the present invention, in its active form, comprisesthree sites for binding to a calcium ion where the calcium ion complexedwith this site constitutes one of the ligands of a negatively chargedphospholipid. The first of these sites, called principle site, involvesresidues 15, 18, 19 and 59 as calcium ligands. The second of thesesites, called secondary site, involves residues 20 and 22 as calciumligands. The third of these sites, which is a low-affinity secondarysite, involves residues 57, 60 and 65 as calcium ligands.

The residues involved overall in the binding to phospholipids areresidues 12, 15, 16, 19, 20, 22, 50, 55, 57, 58, 69, 60 and 65. Thislist includes residues involved in calcium binding, the phospholipidsbeing calcium ligands.

These residues may, of course, be replaced with residues that carry outthe same function with a view to the same result in accordance with thepresent invention.

By way of example, according to the invention, the peptide of formula(I) may advantageously be a peptide sequence chosen from the peptidesequences ID No. 1 to ID No. 10 in the appendix.

The sequence (I) represents the peptides of the present invention intheir shortest functional form. It is clearly understood that thissequence may also comprise, linked to the N-terminal end and/or to theC-terminal end of the sequence (I), one or more amino acids, for examplefrom 1 to 15 amino acids, in general from 1 to 10 amino acids. Mostpreferably, these additional amino acids barely modify the activity ofthe peptides, or not at all, or else improve them.

For example, a small sequence, referred to below as a functionalizationsequence, may be useful in particular for attaching a label to thepeptide, for attaching a molecule for treating diseases to the peptideand/or for attaching said peptide to a support. The length of thisfunctionalization series will be adjusted according to it use. Ofcourse, this sequence will preferably not interfere with the activity ofthe peptides of the present invention. Those skilled in the art will beable to readily adjust the length and the nature of thisfunctionalization sequence according to the use that they will make of apeptide of the present invention.

Thus, according to a first particular embodiment of the presentinvention, the peptides of the present invention may comprise, forexample at their N-terminal end, a functionalization sequence of threeamino acids. This functionalization sequence makes it possible todirectly attach a molecule for treating diseases to the peptide and/orto directly attach said peptide to a support. The peptides in accordancewith this embodiment can be defined by the sequence (II) below: (II)J⁻²-J⁻¹-J⁰-J¹-J²-J³-J⁴-J⁵-J⁶-Z⁷-U⁸-J⁹-J¹⁰-U¹¹-Arg-J¹³-J¹⁴-U¹⁵-Lys-Gly-X¹⁸-Gly-Thr-J²¹-Glu-J²³-J²⁴-U²⁵-J²⁶-J²⁷-J²⁸-U²⁹-J³⁰-J³¹-Arg-J³³-J³⁴-J³⁵-J³⁶-B³⁷-J³⁸-J³⁹-U⁴⁰-J⁴¹-J⁴²-J⁴³-U⁴⁴-J⁴⁵-J⁴⁶-J⁴⁷-J⁴⁸-J⁴⁹-Arg-J⁵¹-U⁵²-J⁵³-J⁵⁴-Asp-U⁵⁶-Lys-Ser-Z⁵⁹-Leu-J⁶¹-J⁶²-J⁶³-J⁶⁴-Z⁶⁵-J⁶⁶-J⁶⁷-U⁶⁸-J⁶⁹-J⁷⁰-J⁷¹-U⁷²- J⁷³-J^(74-J) ⁷⁵in which J, Z, U, X and B are as defined above.

For example, J⁻² may be Gly, J⁻¹ may be Ser, and J⁰ may be Cys, Thr,Pro, Ser or Gln. This sequence J⁻²-J⁻¹-J⁰- may be chosen, for example,from Gly-Ser-Cys-, Gly-Ser-Thr-, Gly-Ser-Pro-, Gly-Ser-Ser-,Gly-Ser-Gly-, and Gly-Ser-Gln-. Thus, for example, each of the sequencesID No. 1 to ID No. 10 mentioned above may comprise, by choice, each oneof the abovementioned functional sequences. The sequence ID No. 12 ofthe sequence listing in the appendix is only a nonlimiting example of asequence (I) according to the present invention comprising, at itsN-terminal end, a functional sequence of three amino acids.

According to a second particular embodiment of the present invention,the peptides of sequence (I) may comprise, for example, at theirN-terminal end, a functionalization sequence of four amino acidsJ⁻³-J⁻²-J⁻¹-J⁰, chosen from Gly-Ser-Gly-Cys-, Gly-Cys-Gly-Ser-,Gly-Ser-Gly-Ser-, Gly-Cys-Gly-Cys- and Gly-Cys-Gly-Ser-. Thisfunctionalization sequence is useful, for example, for direct attachmentof a label such as technetium to the peptide. This embodiment isdisclosed below. Thus, for example, each of the sequences ID No. 1 to IDNo. 10 mentioned above may comprise, by choice, each one of theabovementioned functional sequences. The sequences ID No. 11 of thesequence listing in the appendix (several sequences are grouped togetheras a single one under the name ID No. 11) are merely nonlimitingexamples of sequences (I) according to the present invention comprising,at their N-terminal end, a functional sequence of four amino acids.

According to a third particular embodiment of the present invention, thepeptides of sequence (I) may comprise, for example at their N-terminalend, a functionalization sequence of seven to eleven amino acids. Thisfunctionalization sequence is useful, for example, for direct attachmentof a label such as technetium to the peptide. This embodiment isdisclosed below. Thus, for example, each of the sequences ID No. 1 to IDNo. 10 mentioned above may comprise, by choice, each one of theabovementioned functional sequences. It is also possible to replace thesequence Gly-Ser-Gly-Cys of the sequences ID No. 11 to 14 withGly-Bb1-Gly-Bb2, in which Bb1 and Bb2 are, independently, Cys or Ser.These sequences ID No. 13 and 14 of the sequence listing in the appendix(several sequences are grouped together as a single one under the nameID No. 13 or 14) are merely nonlimiting examples of sequences (I)according to the present invention.

The peptides of the present invention have sufficient affinity forcalcium and are capable of binding reversibly to lipid effectors, and inparticular to those that are negatively charged, such asphosphatidylserines, phosphatidic acids, phosphatidylethanolamines,phosphatidylglycerols, cardiolipins and phosphatidylinositol phosphates.

It is a family of peptides, the main property of which is tospecifically recognize the appearance of lipid signals at the surface ofcell membranes in relation to the normal or pathological functioning oftissues.

The peptides of the present invention can be synthesized by theconventional synthetic processes of organic chemistry or of proteinchemistry, and also by genetic recombination in vivo or in vitro, bygenetic engineering, etc.

Thus, the present invention also relates to a process for producing apeptide according to the invention, said process comprising asolid-phase chemical synthesis of said peptide. The chemical synthesiscan be carried out, for example, with an Applied Biosystems mod. 433Aautomatic peptide synthesizer. It can be carried out, for example, byFmoc chemistry, which uses the fluorenylmethyloxycarbonyl group fortemporary protection of the α-amino function of the amino acids.

The technical elements for carrying out this process of peptidesynthesis are known to those skilled in the art. They are described, forexample, in the book Solid-Phase Organic Synthesis by Kevin Burgess(Editor) Wiley-Interscience; ISBN: 0471318256; (February 2000).

The peptide of the invention may also be produced by geneticrecombination in vivo, for example by means of a process comprising thefollowing steps:

-   -   a) preparing a cDNA comprising a basic sequence encoding said        peptide,    -   b) inserting said cDNA into a suitable expression vector,    -   c) transforming a suitable host cell with said vector into which        the cDNA has been inserted, for replication of the plasmid,    -   d) producing said peptide by translation of said cDNA in said        host cell, and    -   e) recovering the synthesized peptide.

According to the invention, the suitable expression vector and the hostcell are chosen according to the usual techniques for geneticrecombination. The vector may be any one of the plasmids generally usedin this technique, for example a plasmid such as the vector pGEX-2T.Similarly, the cell may be chosen according to the usual techniques; itmay, for example, be E. coli.

When an in vitro genetic recombination technique is used, steps c) andd) of the above process are replaced, respectively, with step c′) forintroducing the vector, into which the cDNA has been inserted, into areaction medium that is suitable for replication of the plasmid, andstep d′) for producing said peptide by Translation of said cDNA in saidsuitable reaction medium. The document R. Jagus and G. S. Beckler (1998)Overview of eukaryotic in vitro translation and expression systems,Current Protocols in Cell Biology 11.1.1-11.1.13., 1989 by John Wiley &Sons, Inc., describes in vitro processes that can be used in the presentinvention.

The present invention also provides a chemical assembly with affinityfor a phospholipid, comprising at least two peptides of the presentinvention, which may be identical or different, said peptides beinglinked to one another. These assemblies can be prepared, for example, byinsertion of a flexible peptide linker, for example polyglycine, betweenthe C-terminal residue of a peptide of the invention and the N-terminalresidue of the second peptide, and so on according to the number ofpeptides placed end to end. This polyglycine linker may have the formula-(Gly)_(n)-, n being a integer ranging from 1 to 12, for example greaterthan 4. According to the invention, at least one of the peptides of theassembly can be a peptide comprising a sequence chosen from thesequences ID No. 1 to 10 of the sequence listing in the appendix.

These assemblies can also be synthesized by conventional syntheticprocesses of organic chemistry or of protein chemistry, and also bygenetic recombination in vivo or in vitro, by genetic engineering, etc,for example by one of the abovementioned processes.

The aim of these assemblies is in particular to increase the affinity ofthe peptides of the present invention for the phospholipid, for examplefor a negatively charged phospholipid.

An assembly of the present invention can be used for three purposes:therapy, research and diagnosis, and there are a great many uses.

The pathologies especially targeted by the present invention are: (i)blood clotting disorders, (ii) apoptotic phenomena subsequent to theaction of chemical compounds, of physical effects such as ionizingradiation, or of biological effects such as those linked to theformation or the necrosis of cancerous tissues, other than the normalphenomena of apoptosis, (iii) inflammatory pathologies, and (iv)disorders associated with the relationship between cells- and theextracellular matrix, and in particular collagen.

The peptides of the present invention also have a considerable advantagecompared with the compounds of the prior art: the reversibility of theirfolding processes, which makes it possible to handle them attemperatures which are higher but compatible with the chemical stabilityof the peptides, for the purposes of chemical modifications with the aimof developing molecules that can be used in imaging and in therapeutics.

In addition, due to their small size, the peptides of the presentinvention can be readily combined with other proteins, either so as toform multifunctional chimeric proteins, or so as to introduce amechanism of regulation by means of effectors other than the signallingphospholipids.

The peptides of the present invention can be used, as such, forproducing a medicinal product that can be used for a treatment or forprophylaxis, since they have intrinsic anticoagulant andanti-inflammatory properties. They make it possible to effect a coatingof cell surfaces, capable of prohibiting the access of compoundsinvolved in the primary steps of blood clotting and inflammatoryphenomena at these surfaces.

Thus, according to the invention, the peptides or assemblies of thepresent invention can be used, as such, for preparing a medicinalproduct, for example chosen from medicinal products intended for thetreatment of a thrombosis, a medicinal product intended for thetreatment of a tumour, and a medicinal product with anti-inflammatoryaction.

The peptides of the present invention can also be used, coupled totreatment molecules, for targeting these; molecules to areas exhibitingnegative charges, such as a thrombus site or a site of inflammation orto an area of tumour. In this application, the peptides and assembliesof the present invention are, for example, coupled respectively to amolecule which has thrombolytic action, to a molecule which hasanti-inflammatory action or to a molecule which has anti-tumour action.Examples of molecules with thrombolytic action that can be usedaccording to the present invention are streptokinases, urokinases andplasminogen activators. In general, the peptides and assemblies of thepresent invention can be coupled, without distinction, to pro-apoptotic,anti-apoptotic and anti-inflammatory compounds.

The peptides and assemblies of the present invention can therefore beused, coupled to a molecule with thrombolytic activity, for producing amedicinal product that can be used in the treatment and the prophylaxisof thrombosis; coupled to a molecule with anti-inflammatory action, forproducing a medicinal product that can be used, for example, locally orintravenously for treating acute pathologies such as asthma, UC, Crohn'sdisease, septic shock, collagen diseases and arthritis; or coupled to amolecule with anti-tumour action, for producing a medicinal product thatcan be used for treating tumours.

For use in research or diagnosis, the peptides of the present inventioncan be coupled to a labelling molecule for detection thereof. Thislabelling molecule may, for example, be a fluorescent molecule,particles of gold or of dense compounds, such as nanoparticles, forelectron microscopy, a radio element, a paramagnetic compound and, ingeneral, one of the labelling molecules commonly used in laboratories.To facilitate certain labelling or binding, this molecule may be linkedto one of the partners of the avidin-biotin system.

According to the invention, the peptides and the chemical assembliesaccording to the invention can be coupled to a labelling molecule so asto form a labelling compound that can be used, for example, for in vivoor in vitro diagnosis.

In fact, the peptides of the present invention can be used for detectingpathologies involving the appearance of negative charges at the surfaceof cells and the release of microvesicules into the blood: for example,clotting disorders, acute inflammatory pathologies, etc., and apoptosis.

They can, for example, be coupled to radio elements with a shorthalf-life, such as technetium or fluorine 18, and can allow “in vivo”detection of the location of thrombotic zones during vascular accidentsof all sorts, in particular apoptotic and inflammatory foci, usingimaging systems.

The peptides of the present invention can also, for example, be coupledto paramagnetic compounds, such as a gadolinium complex, to any contrastagents that can be used in magnetic resonance imaging (MRI), such as,for example, a paramagnetic compound or a ferromagnetic compound, or toany radio active element with a short life time. They can thus allow “invivo” detection of the location of thrombotic zones, and apoptotic andinflammatory zones.

The abovementioned couplings can be carried out by any of theconventional techniques of organic chemistry known to those skilled inthe art.

For example, in the case of technetium, the latter can be coupleddirectly to the peptide of the present invention, for example when thepeptide of sequence (I) comprises a functionalization sequence such asthose described above. This type of coupling is described, for examplein document U.S. Pat. No. 6,323,313 by J. F. Tait et al. Those skilledin the art will understand that labels equivalent to technetium may alsobe coupled, in this way, directly to the peptides of the presentinvention.

Technetium, or any other metal such as those hereby mentioned, can alsobe coupled indirectly to the peptides of the present invention. Thiscoupling is carried out, for example, by means of a cage that complexessaid metal. This cage can be attached to the peptides of the presentinvention by means of a functionalization sequence such as thosedescribed above. In the example of technetium, technetium cages that canbe used according to the present invention are described, for example,in the document 99 mTc Labeling of Highly Potent Small Peptides ShuangLiu, D. Scott Edwards, and John A. Barrett, Bioconjugate. Chem. 1997, 8,621-636.

The peptides or assemblies that are coupled or ready to be coupled,according to the desired application, can be advantageously packaged inthe form of diagnostic kits. Thus, the present invention also provides adiagnostic kit comprising a peptide or an assembly in accordance withthe present invention. This diagnostic kit can, for example, alsocomprise a suitable reagent for detecting said labelling molecule.

The present invention also provides a kit for analysing and detectingnegative charges at the surface of cells, characterized in that itcomprises a peptide or a chemical assembly of the present invention, itbeing possible for the peptide or the assembly to be coupled to a label.

The present invention also provides a kit for analysing and detectingmicrovesicules in the blood, characterized in that it comprises apeptide or a chemical assembly in accordance with the present invention,it being possible for the peptide or the assembly to be coupled to alabel.

In another application of the present invention, peptides or assembliesof the invention can be used, for example, for covering a biocompatiblematerial. This type of material can be used in two types of conditions:i) extracorporeal circulations, and ii) blood storage.

Thus, the peptides of the present invention find an application forexample, in the production of a filter for trapping and recovering, inextracorporeal blood circulation, activated circulating cells:platelets, red blood cells, leucocytes, etc. The blood reintroduced intothe patient will thus be freed of the cells capable of creating abnormalcoagulations, febrile reactions, etc. This filter can be in the form ofa pleated film of biocompatible polymer onto which the peptides of theinvention can be grafted by any appropriate means. These same filterscan be introduced into the bags used to store the blood or can coat theinside of said bags. These filters constitute “sponges” capable ofcontinuously capturing the blood cells containing the bags which areactivated in particular subsequent to them ageing and to them undergoingthe apoptotic process.

The various labellings, couplings and attachments disclosed above willbe most preferably carried out while preserving the activity of thepeptide of the present invention, in general at the ends or in theregion of the ends of the peptides of the present invention or onsurface residues.

Other advantages and characteristics of the present invention willbecome further apparent on reading the nonlimiting illustrative exampleswhich follow, with reference to the figures in the appendix.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The sequences ID No. 1 to ID No. 14 in the appendix are examples ofpeptides comprising the peptide sequence (I) and (II) of the presentinvention.

In particular, the sequences ID No. 11, ID No. 13 and ID No. 14 areexamples of peptides comprising the peptide sequence of the presentinvention into which mutations have been introduced in order to increasethe affinity for calcium and phospholipids.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 are micrographs obtained from tissue sections,respectively, of an apoptotic heart (FIG. 1) and of a kidney (FIG. 2).These sections were obtained, firstly (images on the left) withAFIM-fluorescein (AFIM-F) peptides of the present invention, secondly(images on the right) with annexin 5-fluorescein (A5-F) (compound of theprior art): fluorescence microscopy, magnification ×40. The images inthe centre were obtained with haematoxylin: visible light microscope,magnification ×40. In FIG. 1, the upper and lower photographs representvarious heart sections.

FIG. 3 is a graph which represents the degree of helicity “H” (as %) ofa peptide according to the present invention as a function of thetemperature “t” in ° C.

EXAMPLES Example 1 Synthesis by Genetic Recombination: Expression andPurification of the Peptides of Sequences ID No. 1 to ID No. 12 of thePresent Invention

The sequences ID No. 1 to ID No. 14 were prepared by overexpression inE. coli according to the same protocol as that which was described by F.Cordier-Ochsenbein et al., in J. Mol. Biol. 279, 1177-1185.

The cDNAs of each of these sequences were prepared using a polymerasechain reaction (PCR). They were inserted into the vector pGEX-2T (Smith& Johnson, 1998). FIG. 2 is a diagram illustrating the insertion of thecDNA into the vector. The absence of mutations induced by the PCR wasverified by sequencing.

The production of the peptides is carried out using the E. coli strainBL21 containing the expression vector described above. After inductionwith isopropylthiogalactopyranoside (IPTG, 100 μm) up to an opticaldensity of 1 at 600 nm, the growth is continued until a plateau isreached, i.e. for approximately 3 hours. After centrifugation, thebacteria are resuspended in the lysis buffer comprising 50 mM Tris-HCl,pH 8, 10 mM EDTA, 500 mM NaCl, 5% (v/v) glycerol, 1% (v/v) Triton X100,1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and20 μg/ml of aprotinin.

The purification was carried out in the following way: after sonicationand centrifugation at 10 000 g, the supernatant containing the solubleproteins is incubated with glutathione/agarose beads, allowing specificbinding to these beads of the GST-domain fusion protein. After washingwith a solution containing 1 M NaCl, 50 mM Tris-HCl, at pH 8, 70 unitsof thrombin per litre of culture are added and the sequences are eluted.

The sequences are then purified on a proRPC™ column of 16/10 type,provided by the company Pharmacia, using an FPLC system and at lineargradient of Millipore™ quality water containing 0.1% (v/v) oftrifluoroacetic acid, TFA, and of acetonitrile containing 0.1% of TFA.The flow rate is adjusted to 2.5 ml/minute. Sequences are thenlyophilized.

The final yield for each peptide is approximately 8 mg of sequence perlitre of culture.

Example 2 Example of Chemical Synthesis of Peptides of the PresentInvention

The peptides of the present invention were produced, in this example, bysolid-phase chemical synthesis with an Applied Biosystems mod. 433Aautomatic peptide synthesizer, and with Fmoc chemistry, which uses thefluorenylmethyloxycarbonyl (Fmoc) group for temporary protection of theα-amino function of the amino acids.

The protective groups used to prevent the side reactions of the aminoacid side chains, in this Fmoc strategy, were tert-butyl ether (tBu) forthe Ser, Thr, and Tyr residues; tert-butyl ester (OtBu) for Asp and Glu;trityl (Trt) for Gln, Asn, Cys and His; tert-butyloxycarbonyl (Boc) forLys; and 2,2,5,7,8-pentamethylchromane-6-sulphonyl (Pmc) for Arg.

The coupling reaction is carried out with an excess of 10 equivalents ofamino acid (1 mmol) with respect to the resin (0.1 mmol). The protectedamino acid is dissolved in 1 ml of N-methylpyrrolidone (NMP) and 1 ml ofa 1 M solution of 1-N-hydroxy-7-azabenzotriazole (HOAt) in the solventNMP. 1 ml of a 1 M solution of N,N′-dicyclohexylcarbodiimide (DCC) isthen added. After activation for 40 to 50 minutes, the active esterformed is transferred into the reactor which contains the resin. Beforethe transfer and then coupling step, the resin is deprotected withrespect to its Fmoc group with a 20% solution of piperidine in NMP. Theexcess piperidine is removed by washing with MNP after approximately 5to 10 minutes.

During the deprotection, detection of the dibenzofulvene-piperidineadducts at 305 nm makes it possible to monitor the correct progress ofthe synthesis. In fact, quantification of the adduct makes it possibleto estimate the effectiveness of the deprotection of the Fmoc group and,consequently, of the coupling of the last amino acid incorporated.

The cleavage of the resin and of the protective groups present on theside chains was carried out simultaneously by treatment of the peptidelinked to the resin with trifluoroacetic acid. (TFA). Before carryingout the cleavage, the resin was washed several times withdichloromethane (DCM) and, finally, dried. The reactant used in thecleavage is an acid mixture containing 81.5% of TFA andtriisopropylsilane (1%), ethanedithiol (2.5% when the peptide comprisesa cysteine), water (5%) and phenol scavengers (5%). The resin wastreated with this mixture for three hours with stirring and at ambienttemperature, at a rate of 100 ml of solution per gram of resin. The freepeptide in solution was recovered by filtration. The peptide was thenprecipitated and washed under cold conditions in diisopropyl ether, andthen dissolved in 20% acetic acid and lyophilized.

The peptide recovered after lyophilization, the synthesis cruder is inreduced form, i.e. the interchain disulphide bridges are not formed.

The peptide is then purified on a proRPC™ column type 16/10, provided bythe company Pharmacia, using an FPLC system and a linear gradient ofMillipore™ quality water containing 0.1% by volume of trifluoroaceticacid TFA, and of acetonitrile containing 0.1% of TFA. The flow rate isadjusted to 2.5 ml/minute. The peptide is then lyophilized.

The products obtained were analysed by mass spectrometry.

Example 3 Stability of the Sequences ID No. 1 to ID No. 14

This example shows that the peptides of the present invention constitutestably folded proteins.

Composition of the Blank (Control):

50 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8 10 μl

H₂O 990 μl

Adjusted to pH 8.

Composition of the Sample:

Sample: domain purified in 50 mM Tris buffer containing 150 mM NaCl, pH8. Approx. concentration: 200 mg/ml.

Domain: 10 μl, i.e. final concentration of 300 μM.

H₂O: 990 μl.

pH measured at 7.8.

Hardware and Software Configuration:

Jobin Yvon CD6 device

CD-max software

Optical path of the measuring cuvette: 1 cm.

FIG. 1 in the appendix represents the degree of helicity of AFIM as afunction of the temperature, as measured using the circular dichroismasignal in the far-UV at the wavelength of 200 nm.

In this figure, the value of the signal at 14° C. is taken for 100% ofthe helical content of the peptide.

The thermal denaturation of the peptide is clearly cooperative anddemonstrates that, at low temperature, and in particular at 37° C., itis a peptide that is suitably folded and exhibits improved stability.

Example 4 Assemblies of Two Peptides of the Present Invention

The process described in example 1 above is used to synthesize a peptidesequence of sequence ID No. 1-(Gly)₄—ID No. 1.

The final yield for the assembly is approximately 14 mg/litre ofculture.

Example 5 Fluorescein Labelling of a Peptide of the Present Invention

In the examples which follow, the peptide of the present invention iscalled AFIM-SH. It has a peptide sequence as defined by the sequence(I). The sequences ID No. 1 to ID No. 14 are tested.

Fluorescein is a molecule which emits a green fluorescence with awavelength of 525 nm when it is excited at a wavelength of 488 nm. Theemission of green light is detected by cameras or photomultipliers. Thiscoupling of AFIM to fluorescein makes it possible to detect the presenceof cells exhibiting PS both in vitro and in vivo in small animals.

According to the present invention, it is possible to label AFIM on thesurface residues, on any cysteine which would be introduced in place ofany amino acid present at the surface of AFIM (surface residues)provided that the lipid membrane-binding function is not disturbed. AFIMthus modified is referred to as AFIM-SH below.

The coupling of the fluorescein is carried out by means of a maleimidefunction represented below on AFIM by the function SH.

The fluorescein is coupled to one or more cysteine(s) of the sequence,covalently, using a maleimide function.

All the labelling is carried out at a temperature below 20° C.

AFIM-SH is in solution in Tris buffer (50 mM) containing NaCl (150 mM),pH=7.4. 5 equivalents of DTT in solution in the same buffer are added tothe AFIM-SH solution. The medium is stirred for 30 min.

In the dark: fluorescein (5 equivalents of AFIM-SH+2 equivalents of DTT)is weighed out and dissolved in DMF, and added to the above solution.The entire mixture is stirred and the reaction is continued for 30 min.The medium is then diluted in 150 ml of PBS buffer (20 mM phosphate, 150mM NaCl), pH=7.4, and ultrafiltered through a YM3 membrane™. The sampleis re-diluted and ultrafiltered several times, recording the UV spectrumof the filtrate.

When there is no more fluorescein in the filtrate (peak at 490 nm), thesample is concentrated to a few ml and stored chilled at 4° C.

The AFIM-fluorescein products were used to detect apoptotic cells byflow cytometry in vitro, and also in animals in vivo, in the mannerdescribed in example 6 below.

Example 6 Results of Labelling of Apoptotic Cells with theAFIM-Fluorescein Products

Imaging of apoptotic cardiac cells after infarction in rats.

A model of apoptosis in rats is used as described in the articlepublished in Circulation Res. 1996, 79, 946-956.

Briefly, four rats (each weighing 300 g) were anaesthetized, intubatedand ventilated. The myocardial ischemia was caused by temporaryocclusion of the coronary artery. After occlusion for 30 minutes, thecoronary artery was re-perfused for one hour.

At the end of the re-perfusion period, the AFIM-fluorescein peptides ofexample 5 were injected in the jugular vein at a rate of 200 μg ofpeptide for each of: two of the rats in the total volume of 1 ml.

By way of comparison, 200 μg of annexin 5-fluorescein (compounds of theprior art) were injected under the same conditions for each of the othertwo rats in a total volume of 1 ml.

The rats were sacrificed after 60 minutes.

Five organs were conserved for this study: the heart, the lung, thekidney, the liver and the brain. They were washed and rinsed in thepresence of formol. The organs were then dehydrated and impregnated withparaffin for approximately 12 hours and then 7 μm sections were cut.

Some sections were stained with haematoxylin. The sections were examinedunder a fluorescence microscope and the adjacent sections stained withhaematoxylin were examined with a visible light microscope. Thehaematoxylin-stained sections (marked H1 and H2 respectively on FIGS. 1and 2 in the appendix) allow the architecture of the tissue to bevisualized and the fluorescence microscopy makes it possible to detectthe labelling with AFIM-fluorescein (AFIM-F) or with annexin5-fluorescein (A5-F).

FIG. 1 in the appendix shows the images obtained for the apoptotic heartand FIG. 2 in the appendix shows the images obtained for the kidney.

FIG. 1 clearly shows the excess of fluorescein corresponding to theaccumulation of label in the apoptotic cells. The contrast is visiblymuch better with AFIM of the present invention than with annexin 5 ofthe prior art.

FIG. 2 shows the labelling of the kidney associate with the partialelimination of the product. In the case of AFIM, the glomeruli do notappear to be labelled, only the proximal tubules are partially labelled.On the other hand, in the case of annexin 5 of the prior art, the entirerenal tissue is strongly labelled, which is in agreement with the renaltoxicity observed for this protein.

The results obtained in this example demonstrate a great specificity ofthe peptides of the present invention for cell labelling.

Labelling of the AFIM peptide, for example of ID No. 1 to 10, withfluorescein therefore makes it possible to effectively detect thephosphatidylserine (PS) present at the outer surface of cells involvedin physiopathological processes such as programmed cell death(apoptosis), blood clotting or inflammatory reaction.

Example 7 Labelling of a Peptide of the Present Invention withTechnetium ^(99m)Tc

The labelling of AFIM with ^(99m)Tc makes it possible, as forfluorescein, to detect phosphatidylserine (PS) present at the outersurface of cells involved in physiopathological processes such asprogrammed cell death (apoptosis), blood clotting or inflammatoryreaction. ^(99m)Tc is a γ-ray emitter which makes it possible to detectAFIM in any region of the body by means of various types of cameras thatdetect γ-radiation. This coupling of AFIM to ^(99m)Tc makes it possibleto detect the presence of cells exhibiting PS in vivo in any livingbeing.

Two types of technetium labelling are disclosed in this example:indirect labelling (A) and direct labelling (B).

A) Indirect Labelling

In this example, AFIM-SH is coupled, at a cysteine in its sequence, to acomplexing molecule, called cage molecule, capable of specificallyreceiving the ^(99m)Tc ion. The coupling reaction is representedschematically below (Scheme II).

The cage molecule chosen is NH₂—C₃(Bham)₂ (2) described in the followingdocument: Bis(Hydroxamamide)-Based Bifunctional Chelating Agent for^(99m)Tc Labelling of Polypeptides, Le-Cun Xu et al. Biconjugate Chem.1999, 10, 9-17. This cage is coupled to the maleimide derivative (1)according to Scheme (II) so as to give the label (3) which is thencoupled to AFIM-SH so as to give the compound referred to as AFIM-cage(Scheme II)). The coupling is carried out in the following way:

AFIM-SH is in solution in Tris buffer (50 mM) containing NaCl (150 mM),pH=7.4. 5 equivalents of tris-(2-carboxyethyl)phosphine (TCEP)hydrochloride are weighed out dissolved in the same buffer, and added toAFIM-SH. The medium is stirred and left at ambient temperature for 3.0minutes.

During this time, 10 equivalents of (1) are dissolved indimethylformamide (DMF) and transferred onto 20 equivalents of cage (2)in the same volume of DMF. After reaction for 10 min, the product isadded to AFIM-SH.

The entire mixture is stirred, and the reaction is continued at ambienttemperature for 30 min. The medium is then dissolved in 150 ml of Trisbuffer (50 mM) containing NaCl (150 ml), pH=7.4, and ultrafilteredthrough a YM3 membrane™. The same is re-diluted and ultrafilteredseveral times, recording the UV spectrum of the filtrate. When there isno more DMF in the filtrate (peak at 214 nm), the sample is concentratedto a few ml and stored chilled (4° C.).

An amount suitable for the size of the animal, of the peptide coupled tothe technetium cage (AFIM-cage) prepared in this example, is taken andan aqueous solution of SnCl₂ (6 equivalents relative to the peptide) isadded. The ^(99m)TcO₄ ⁻ solution is added and the reaction is continuedfor 30 minutes at ambient temperature.

The solution of labelled peptide (AFIM-cage-^(99m)Tc) is then directlyinjected intravenously into the animal.

The images are then collected by means of a camera capable of detectingγ-rays (SPECT or other camera).

B) Direct Labelling

In this example, AFIM is labelled with technetium without a cage. Forthis, AFIM is provided with a functionalization sequence of four aminoacids which directly bind the technetium.

The peptide sequence ID No. 11 is used in this example. Thefunctionalization sequence is Gly-Ser-Gly-Cys on the N-terminal side,residues 5 to 79 of the sequence ID No. 11 being those forming thesequence (I) of the present invention.

For the labelling, the peptide of sequence ID No, 11 and 5 equivalentsof TCEP are dissolved in physiological saline and equilibrated for 15min. 10 equivalents of SnCl₂ are then added. This solution can belyophilized and stored under nitrogen.

The labelling is carried out by adding a solution of ^(99m)TcO₄ ⁻. Afterincubation for 15 minutes, the solution is passed over a PD10™ column.

The sequence ID No. 11 directly labelled with technetium (AFIM-^(99m)Tc)is injected intravenously.

The images are then collected with a camera such as those used above.

Example 8 Gadolinium Labelling of a Peptide of the Present Invention

AFIM coupled to gadolinium: AFIM-cage-Gd (indirect labelling)

The gadolinium labelling of AFIM makes it possible, as for the previouslabels, to detect the phosphatidylserine (PS) present at the outersurface of cells involved in physiopathological processes such asprogrammed cell death (apoptosis), blood clotting or inflammatoryreaction. Gadolinium is a paramagnetic agent which makes it possible todetect AFIM in any region of the body by means of nuclear magneticresonance imaging processes. This coupling of AFIM to gadolinium makesit possible to detect, with a resolution which can range up to 1 mm, thepresence of cells exhibiting PS in vivo in any living being.

As for fluorescein, AFIM can be coupled, at a cysteine, to a chemicalmolecule capable of specifically receiving the gadolinium ion. Once thisgadolinium cage has been constructed, the coupling is carried out withAFIM as described below.

AFIM-SH is in solution in Tris buffer (50 mM) containing NaCl (150 mM),pH=7.4. 5 equivalents of TCEP are weighed out and dissolved in the samebuffer, and added to AFIM-SH. The medium is stirred and left at ambienttemperature for 30 min.

The gadolinium cage used is that described in the document P. KANTHI etal., “Synthesis of Charged and Uncharged Complexes of Gadolinium andYttrium with Cyclic Polyazaphosphinic Acid Ligands for in vivoApplications”, J. CHEM SOC. PEKIN TRANS. 2, 1993, pp. 605-618.

5 equivalents of cage, relative to AFIM-SH, are dissolved in DMF andadded to AFIM-SH. The entire mixture is stirred, and the reaction iscontinued for 30 min at ambient temperature. The medium is thendissolved in 150 ml of Tris buffer (50 mM) containing NaCl (150 nM),pH=7.4, and ultrafiltered through a YM3 membrane. The sample isre-diluted and ultrafiltered several times, recording the UV spectrum ofthe filtrate. When there is no more DMF in the filtrate (peak at 214nm), the sample is concentrated to a few ml and stored chilled at 4° C.

Once purified, the product is injected intravenously.

The images are collected by means of a detection camera.

Example 9 Gold Labelling of a Peptide of the Present Invention

The labelling of AFIM with gold is a direct labelling.

It makes it possible to detect the phosphtidylserine (PS) present at theouter surface of the cells involved in physiopathological processes suchas programmed cell death (apoptosis), blood clotting or inflammatoryreaction.

Gold is an electron-dense metal, which means that it can be used inelectron microscopy. This coupling of AFIM to gold makes it possible todetect and to locate the phosphatidylserine on a cellular andsubcellular compartment scale. The coupled product can be used in vitro.

AFIM-SH is dissolved in Tris buffer (50 mM) containing NaCl (150 mM),pH=7.4. 5 equivalents of tris-(2-carboxyethyl)phosphine (TCEP) insolution in the same buffer are added to AFIM-SH. The medium is stirredfor 15 min.

Modified gold beads (containing a grafted maleimide: NanogoldMonomaleimide Interchim™) are dissolved in 20 μl of DMSO and 200 μl ofwater, and added to the above solution (2 equivalents of beads relativeto the protein).

The entire mixture is stirred, and the reaction is continued for onehour. The medium is then purified on a gel filtration column (PharmaciaPD-10™) and eluted with PBS buffer (20 mM phosphate, 150 mM NaCl),pH=7.4.

AFIM-Au can be used on tissue sections or on isolated cells. Theanalysis can be carried out by electron microscopy.

1. Peptide consisting of the peptide sequence (I) below: (I; SEQ ID NO:15) J¹-J²-J³-J⁴-J⁵-J⁶-Z⁷-U⁸-J⁹-J¹⁰-U¹¹-Arg-J¹³-J¹⁴-U¹⁵-Lys-Gly-X¹⁸-Gly-Thr-J²¹-Glu-J²³-J²⁴-U²⁵-J²⁶-J²⁷-J²⁸-U²⁹-J³⁰-J³¹-Arg-J³³-J³⁴-J³⁵-J³⁶-B³⁷-J³⁸-J³⁹-U⁴⁰-J⁴¹-J⁴²-J⁴³-U⁴⁴-J⁴⁵-J⁴⁶-J⁴⁷-J⁴⁸-J⁴⁹-Arg-J⁵¹-U⁵²-J⁵³-J⁵⁴-Asp-U⁵⁶-Lys-Ser-Z⁵⁹-Leu-J⁶¹-J⁶²-J⁶³-J⁶⁴-Z⁶⁵-J⁶⁶-J⁶⁷-U⁶⁸-J⁶⁹-J⁷⁰-J⁷¹-U⁷²-J⁷³-J⁷⁴- J⁷⁵

in which J, Z, U, X and B represent amino acids such that: the aminoacids J are chosen, independently of one another, from natural aminoacids or derivatives thereof, such that at least 50% of them are polarresidues chosen from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Lys, Orn,Pro, Ser, Thr and Tyr, the amino acids U are chosen from Ala, Cys, Gly,Ile, Leu, Met, Phe, Trp, Tyr and Val, the amino acid X¹⁸ is chosen,independently of the other amino acids of the sequence, from Ala, Asn,Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Ser, Thr, Trp, Tyr and Val, theamino acid B³⁷ is chosen, independently of the other amino acids of thesequence, from Arg, Ala, Cys, Gly, Ile, Leu, Met, Phe, Trp, Tyr and Val,the amino acid Z⁷ is chosen, independently of the other amino acids ofthe sequence, from Asp and Glu, the amino acids Z⁵⁹ and Z⁶⁵ are chosen,independently, from Glu, Asp, Lys or Arg, the superscripts of J, Z, U, Xand B representing the position of these amino acids in said
 2. Peptideaccording to claim 1, in which the amino acids J are chosen,independently of one another, from Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, suchthat at least 50% of them are polar residues chosen from Arg, Asn, Asp,Gln, Glu, Gly, His, Lys, Pro, Ser and Thr.
 3. Peptide according to claim1, in which the amino acids U and B of the sequence (I) are chosenaccording to one of the examples a) to j) disclosed in table 1 below: U⁸U¹¹ U¹⁵ U²⁵ U²⁹ B³⁷ U⁴⁰ U⁴⁴ U⁵² U⁵⁶ U⁶⁸ U⁷² Ex a) Val Leu Met Ile LeuArg Ile Tyr Leu Leu Val Leu Ex b) Ala Ile Ile Ile Leu Arg Ile Tyr LeuLeu Ile Leu Ex c) Ala Ile Ile Ile Leu Arg Ile Tyr Leu Leu Met Val Ex d)Ala Leu Met Leu Leu Arg Ile Tyr Leu Leu Ile Met Ex e) Ala Leu Met IleIle Arg Val Tyr Leu Leu Ile Met Ex f) Ala Leu Met Ile Ile Arg Ile PheLeu Leu Ile Met Ex g) Ala Leu Met Ile Val Arg Ile Phe Leu Leu Ile Phe Exh) Val Leu Met Ile Leu Arg Ile Phe Leu Leu Ile Met Ex i) Ala Leu Met IleLeu Arg Ile Phe Leu Leu Ile Met Ex j) Ala Leu Met Ile Leu Arg Ile TyrLeu Leu Ala Ala Ex k) Val Leu Met Ile Leu Arg Ile Tyr Leu Leu Val Leu Exl) Val Leu Met Ile Leu Arg Ile Phe Leu Leu Val Leu(Ex = example)


4. Peptide consisting of a sequence chosen from the sequences of SEQ IDNo. 1 to SEQ ID No.
 10. 5. Peptide consisting of the sequence of SEQ IDNo.
 1. 6. Peptide according to claim 1, wherein a tripeptide sequence islinked to the N-terminal end of the sequence (I), wherein saidtripeptide sequence is selected from the grout consisting ofGly-Ser-Cys-, Gly-Ser-Thr-, Gly-Ser-Pro-, Gly-Ser-Ser-, Gly-Ser-Gly-,and Gly-Ser-Gln-.
 7. Peptide according to claim 1, wherein atetrapeptide sequence is linked to the N-terminal end of the sequence(I), wherein said tetrapeptide sequence is selected from the groupconsisting of Gly-Ser-Gly-Cys- (SEQ ID NO: 17), Gly-Cys-Gly-Ser- (SEQ IDNO: 18), Gly-Ser-Gly-Ser- (SEQ ID NO: 19), and Gly-Cys-Gly-Cys- (SEQ IDNO: 20).
 8. Peptide consisting of the sequence of SEQ ID No. 11 or SEQID No.
 12. 9. Peptide consisting of the sequence of SEQ ID No. 13 or SEQID No.
 14. 10. Process for producing a peptide according to claim 1,said process comprising solid-phase chemical synthesis of said peptide.11. Process for producing a peptide according to claim 1, in culture,said process comprising: a) preparing a cDNA comprising a basic sequenceencoding said peptide, b) inserting said cDNA into a suitable expressionvector, c) transforming a suitable host cell with said vector into whichthe cDNA has been inserted, for replication of the plasmid, d) producingsaid peptide by translation of said cDNA in said host cell, and e)recovering the synthesized peptide.
 12. Process according to claim 11,in which the vector is a plasmid.
 13. Process according to claim 11, inwhich the vector is the vector pGEX-2T.
 14. Process according to claim11, in which the host cell is E. coli.
 15. Chemical assembly withaffinity for a phospholipid, comprising at least two peptides as definedin claim 1, which may be identical or different, said peptides beinglinked to one another.
 16. Chemical assembly according to claim 15, inwhich at least one of the peptides is a peptide consisting of a sequencechosen from the sequences of SEQ ID No. 1 to SEQ ID No.
 10. 17. A methodfor covering a biomaterial comprising contacting said biomaterial with apeptide according to claim
 1. 18. A method for producing a filter fortrapping activated circulating blood cells immobilizing a peptideaccording to claim 1 said filter.
 19. Labelling compound comprising apeptide as defined in claim 1, coupled to a labelling molecule or tonanoparticles that are dense in electron microscopy.
 20. Labellingcompound comprising an assembly as defined in claim 15 coupled to alabelling molecule or to nanoparticles that are dense in electronmicroscopy.
 21. Compound according to claim 19, in which the labellingmolecule is a fluorescent molecule.
 22. Compound according to claim 19,in which the labelling molecule consists of one of the partners of theavidin-biotin system.
 23. Compound according to claim 19, in which thelabelling molecule is a radio element.
 24. Compound according to claim19, in which the labelling molecule is a contrast agent in magneticresonance imaging.
 25. Compound according to claim 19, in which thelabelling molecule is technetium.
 26. Compound according to claim 19, inwhich the nanoparticles that are dense in electron microscopy are goldnanoparticles.
 27. Diagnostic kit comprising a compound according toclaim
 19. 28. Diagnostic kit according to claim 27, also comprising asuitable reagent for detecting said labelling molecule.
 29. Kit foranalysing and detecting negative charges at the surface of cells,characterized in that it comprises a peptide according to claim
 1. 30.Kit for analysing and detecting negative charges at the surface ofcells, characterized in that it comprises an assembly according to claim15.
 31. Kit for analysing and detecting microvesicules in the blood,characterized in that it comprises a peptide according to claim
 1. 32.Kit for analysing and detecting microvesicules in the blood,characterized in that it comprises an assembly according to claim 15.33. Kit according to claim 29, in which the peptide is coupled to alabel.
 34. Kit according to claim 30, in which the assembly is coupledto a label.
 35. Filter for dialysing activated circulating blood cells,said filter being characterized in that it comprises a peptide accordingto claim
 1. 36. Peptide according to claim 4, wherein a tripeptidesequence is linked to the N-terminal end of the sequence (I), whereinsaid tripeptide sequence is selected from the group consisting ofGly-Ser-Cys-, Gly-Ser-Thr-, Gly-Ser-Pro-, Gly-Ser-Ser-, Gly-Ser-Gly-,and Gly-Ser-Gln-.
 37. Peptide according to claim 4, wherein atetrapeptide sequence is linked to the N-terminal end of the sequence(I), wherein said tetrapeptide sequence is selected from the groupconsisting of Gly-Ser-Gly-Cys-, Gly-Cys-Gly-Ser-, Gly-Ser-Gly-Ser-,Gly-Cys-Gly-Cys- or Gly-Cys-Gly-Ser-.
 38. Peptide according to claim 5,wherein a tripeptide sequence is linked to the N-terminal end of thesequence (I), wherein said tripeptide sequence is selected from thegroup consisting of Gly-Ser-Cys-, Gly-Ser-Thr-, Gly-Ser-Pro-,Gly-Ser-Ser-, Gly-Ser-Gly-, and Gly-Ser-Gln-.
 39. Peptide according toclaim 5, wherein a tetrapeptide sequence is linked to the N-terminal endof the sequence (I), wherein said tetrapeptide sequence is selected fromthe group consisting of Gly-Ser-Gly-Cys-, Gly-Cys-Gly-Ser-,Gly-Ser-Gly-Ser-, Gly-Cys-Gly-Cys- or Gly-Cys-Gly-Ser-.
 40. Labellingcompound comprising an assembly as defined in claim 16, coupled to alabelling molecule or to nanoparticles that are dense in electronmicroscopy.
 41. Compound according to claim 40, in which the labellingmolecule is a fluorescent molecule.
 42. Compound according to claim 40,in which the labelling molecule consists of one of the partners of theavidin-biotin system.
 43. Compound according to claim 40, in which thelabelling molecule is a radio element.
 44. Compound according to claim40, in which the labelling molecule is a contrast agent in magneticresonance imaging.
 45. Compound according to claim 40, in which thelabelling molecule is technetium.
 46. Compound according to claim 40, inwhich the nanoparticles that are dense in electron microscopy are goldnanoparticles.
 47. Diagnostic kit comprising a compound according toclaim
 40. 48. Diagnostic kit according to claim 47, also comprising asuitable reagent for detecting said labelling molecule.
 49. Kit foranalysing and detecting negative charges at the surface of cells,characterized in that it comprises an assembly according to claim 16.50. Kit for analysing and detecting microvesicules in the blood,characterized in that it comprises an assembly according to claim 16.51. Kit according to claim 50, in which the assembly is coupled to alabel.
 52. Compound according to claim 20, in which the labellingmolecule is a fluorescent molecule.
 53. Compound according to claim 20,in which the labelling molecule consists of one of the partners of theavidin-biotin system.
 54. Compound according to claim 20, in which thelabelling molecule is a radio element.
 55. Compound according to claim20, in which the labelling molecule is a contrast agent in magneticresonance imaging.
 56. Compound according to claim 20, in which thelabelling molecule is technetium.
 57. Compound according to claim 20, inwhich the nanoparticles that are dense in electron microscopy are goldnanoparticles.
 58. Diagnostic kit comprising a compound according toclaim
 20. 59. Diagnostic kit according to claim 58, also comprising asuitable reagent for detecting said labelling molecule.
 60. Kitaccording to claim 31, in which the peptide is coupled to a label. 61.Kit according to claim 32, in which the assembly is coupled to a label.